Putative mitochondrial polypeptides coded by expanded quadruplet codons, decoded by antisense tRNAs with unusual anticodons

Weak triplet codon–anticodon interactions render ribosome-free translation unlikely. Some modern tRNAs read quadruplet codons (tetracodons), suggesting vestigial ribosome-free translation. Here, mitochondrial genomes are explored for tetracoded overlapping protein coding (tetra)genes. Occasional single tetracodons within regular mitochondrial genes coevolve positively/negatively with antisense tRNAs with predicted reduced/expanded anticodons (depending on taxon), suggesting complex tetra-decoding mechanisms. Transcripts of antisense tRNAs with unusual anticodons are more abundant than of homologues with regular anticodons. Assuming overlapping tetracoding with silent 4th tetracodon position, BLAST aligns 10 putative tetragenes spanning 17% of regular human mitochondrial protein coding tricodons with 14 GenBank proteins. Various tests including predicted peptide secondary structures, 3rd codon position (of the regular main frame of the protein coding gene) conservation against replicational deamination mutation gradients, and circular code usage (overlapping genes avoid using circular code codons) confirm tetracoding in these overlapping tetragenes with silent 4th position, but not for BLAST-predicted tetragenes assuming silent 2nd or 3rd positions. This converges with tetradecoding mechanisms that are more compatible with silent 4th, than at other, tetracodon positions. Tetracoding increases with (a) GC-contents, perhaps conserved or switched on in high temperature conditions; (b) usage of theoretically predicted ‘tessera’ tetracodons; (c) 12s rRNA stability; and d) antisense tRNA numbers with predicted expanded anticodons. Most detected tetragenes are not evolutionarily conserved, apparently reflect specific, transient adaptations. Tetracoding increases with mammal longevity.